An episomal marking system for tracking cell proliferation in development, tissue homeostasis, and aging. Abstract Most adult tissues are maintained in a homeostatic state through replacement of aged or damaged cells with young cells derived from adult tissue stem cells. Because stem cells are present in small numbers, they need to expand following each step of differentiation in order to constitute a large tissue mass. Therefore cell proliferation in adult tissues can often be used as an indicator of stem cell activity. It has been generally observed that aging is accompanied with a decline in stem cell activity. For example, studies in mouse models showed that the ailing immune response in aged mice is correlated with a decline in expansion capacity of hematopoietic stem cells and the lymphoid progenitor cells. Thus far, the methods available to assess homeostatic proliferation due to stem cell activity in live animals are still limited. We propose to establish an episomal marking system for tracking cell proliferation in mice. We will use a Cre/lox recombination system to induce and track tissue and stage specific formation of excision circles from a recombination cassette. The recombination event, initiated by Cre recombinase, will lead to simultaneous activation of two visible markers, one on the chromosome and the other on the excision circle. The chromosomal marker will label all the descendant cells. The marked episomal DNA, which does not replicate, will be diluted out following each cell division. The ratio of cells expressing both markers vs. cells expressing only the chromosomal marker provides a simple and quantitative readout of the proliferative history of the population. We plan to establish reporter mice carrying the recombination cassette and test the feasibility of the reporter system by tracking lymphocyte and lymphoid progenitor proliferation during development, homeostasis, and aging. While the proposed study focuses on the lymphoid system, the reporter mice should be generally applicable for studying development and aging in most other tissue types. PUBLIC HEALTH RELEVANCE: The proposed study will establish a new method for tracking ageing related biological changes in mouse models. The experimental system can be used in revealing and investigating genetic and environmental factors that influence ageing.